In the food sector it is becoming increasingly necessary to guarantee greater safety in terms of hygiene and health of food and water.
Both high technology countries and the developing countries, for different reasons, require simple, rapid and inexpensive microbiological control of food and water. So far no microbiological analysis methods with characteristics combining simplicity of use, rapidity of analysis and low cost are available on the market.
Food and water analysis most frequently involves identification of the so-called “indicators”, i.e. micro-organisms which, when absent, provide a reliable indication of the absence of pathogenic micro-organisms. Among the “indicators”, the most important is the quantitative search for total micro-organisms, coliforms/E. coli and coagulase-positive staphylococci (St. aureus). The detection of such micro-organisms covers approximately 40-50% of the microbiological analyses in the water and food sector.
The traditional method for detection of micro-organisms present in a liquid (e.g. water) or in a solid (foodstuffs) is based on microbial multiplication detection. In fact micro-organisms, normally invisible to the naked eye, can be detected when, as a result of multiplication by successive divisions from a single cell (clones), aggregates of billions of cells (colonies) form, visible to the naked eye. The traditional methods based on bacterial multiplication are therefore also called Colony-Forming Unit (CFU) count. Specific detection of a particular type of micro-organism is ensured by the use of selective nutritional media. The multiplication of the micro-organisms can be easily observed by the naked eye in selective media, both solid (plate count method for microbiological examination of liquids) and liquid (method of the “most probable number” for the microbiological examination of solids). In the plate count method, the solid selective media in general are contained in transparent capsules. One drop of the liquid sample to be examined (concentrated on filters if necessary) is deposited on the selective nutritional medium and multiplication of the micro-organisms becomes visible in the form of “colonies”, each formed of billions of cells, all deriving from at least 20-22 successive divisions of one single cell (clones). These clones appear as small protuberances with dimensions from 0.5 to 1 mm or more. From the number of colonies present in one single capsule, the number of micro-organisms initially present in the drop of liquid sample deposited on the nutritional medium can therefore be traced.
In the method of the “most probable number” the liquid selective media are contained in transparent test tubes. A small homogenised quantity of the solid sample to be examined is added to these test tubes. Multiplication of the micro-organisms is indicated by the appearance of a widespread cloudiness of the nutritional medium contained in the test tube. By means of tables drawn up on a statistical basis, the “most probable number” of micro-organisms initially present in the solid sample can be traced according to the presence or absence of the cloudiness in the different test tubes at different dilutions of the sample to be examined.
It is evident from the above that these methods are demanding in terms of the work process and require the presence of an equipped laboratory in order to previously sterilise the material to be used and maintain it in sterile conditions. The absence of sterility would lead to microbial contamination from outside the sample, thus rendering the analysis performed meaningless.
Alternative methods are currently used, such as the one based on the use of chromogenic media. Although this too is a cultivation method, the use of chromogenic media for the microbiological analysis can often be considered a “rapid” method, as it permits optimisation of the search for specific micro-organisms without the need to perform sub-cultures and sometimes even without the need for confirmation tests.
Of the rapid methods, the use of antibodies is the one with the greatest impact in the food sector. The specificity of the monoclonal antibodies, the simplicity and versatility of the antigen-antibody reaction has permitted the development of numerous immunological analysis methods. These methods, however, suffer from the drawbacks relative to the need for specialist personnel and equipment, and the high sensitivity level of at least 104 cells/ml.
Lastly, genetic molecular analysis methods have been used more recently. These methods comprise DNA/DNA hybridisation, analyses of the rRNA (ribosomal RNA) sequences, use of oligonucleotide probes complementary to the rRNA or to other target genes, ribotypification and PCR-Polymerase Chain Reaction. These methods, although they have the advantage of a low sensitivity limit (<102 cells/ml), do not solve the problem of the need for highly specialised personnel for the use of complex equipment, controlled environments to prevent contamination of the sample and, furthermore, are often not able to distinguish between living or dead micro-organisms.